Four independent hematopoietic precursor cell types are specified during development, culminating in specification of hematopoietic stem cells (HSCs), which self-renew and provide all of the major blood lineages over the lifetime of an adult organism. The first three precursor cell types are transient progenitors believed to temporarily provide blood and immune cells to the embryo until HSCs finally emerge. Precisely where and when HSCs are specified has until recently been highly controversial, but recent studies have conclusively demonstrated that they arise from hemogenic endothelium, a special population of endothelial cells within the ventral wall of the primitive dorsal aorta that transdifferentiate into HSCs. In studies performed during our first funding period, we have directly imaged HSC birth from ventral aortic endothelium for the first time. Complementary lineage tracing of these HSC founders indicate that they provide all adult hematopoietic cells, and are thus the unique source of all HSCs. The signaling events underlying the developmental specification of hemogenic endothelium remain poorly understood. In this application, we will utilize the unique experimental advantages aforded by the zebrafish embryo to test and refine a novel model of HSC induction. Our preliminary results suggest that Notch signaling is required at least twice to specify hemogenic endothelium. A first requirement occurs early during somitogenesis and is non-cell autonomous with respect to HSC precursors. We have discovered that Wnt signaling lies upstream of this Notch requirement by regulating the somitic expression of two Notch ligand genes, deltaC and deltaD. How these somitic signaling events relate to the specification of HSC precursors is presently unclear, and form a major research direction of this application. Our studies will begin mechanistically, with a thorough dissection of how the Wnt16 signal is received and transduced, and how these events connect to the regulation of deltaC and deltaD. Next, we will determine which Notch receptor(s) are downstream of DeltaC and DeltaD, and which cell types receive this signal to relay instructive cues to aortic endothelium. Similarly, we will work to further dissect and distinguish the roles of environmental and intrinsic Notch signaling required to specify HSCs. Finally, we will complement our genetic approaches with efforts to better understand the cellular interactions and migration events that relay the Wnt16-Notch dependent signals to pattern the embryonic HSC niche. Our discovery of the Wnt16-Notch pathway represents one of the earliest known environmental regulators of HSC fate. With the elucidation of this signaling axis, and the cellular behaviors it controls, our work will ultimately enable ex vivo approaches to direct patient-specific iPS cells towards the HSC fate for cellular replacement therapies. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells (HSCs) are rare cells within human bone marrow that are responsible both for the life-long replenishment of all blood cell lineages and for the curative effects of bone marrow transplantation. The creation of human induced pluripotent cells holds great promise for cellular regeneration therapies, but we cannot currently instruct these cells to specifically generate HSCs in vitro. The overall goal of this application is to determine the molecular cues that instruct HSC fate in the vertebrate embryo such that these events may ultimately be replicated in vitro for clinical utility.